Reading the cell’s compass to navigate cancer

Imagine for a moment that you couldn’t tell your front from your back or your head from your toes. It would clearly be more than a little difficult to navigate your way around the world.

Knowing which way is up and which is down is also crucial for cells in our body. Lots of daily tasks within cells depend on molecules being sent to either the top or bottom of the cell, and cells need to be aware of which end is which.

The process of setting up and maintaining this innate ‘compass’ is known as cell polarity, and is a hot topic in cancer research.

Our scientists are among those leading the work to uncover how cells control polarity and find out how forgetting which way is up can lead to the development of cancer.

One molecule that is receiving a lot of attention is protein kinase C iota (PKCι), which has a key role in maintaining the accuracy of a cell’s compass.

We discuss their fascinating research and what this means for a cell’s internal compass, both on a day-to-day basis and also in cancer.

Protein origami

Scientists are setting their sights on PKCι because research has revealed that it’s found at unusually high levels in some cancer cells, suggesting it’s playing an important role in the disease. This makes it really important that we understand all the little details about how PKCι works.

Proteins are made up of a string of building blocks called amino acids. Each amino acid can interact with its neighbours, and these interactions combine to push, pull and fold the protein into shape.

This process of molecular origami is crucial, as the shape and structure of a protein dictates what it can and can’t do in a cell. It also determines what other molecules stick to the protein, so finding out the unique shape of proteins is important for understanding how they work.

Sticky patch

Looking how a protein folds can tell us how it works

To see how PKCι folds up into the molecular equivalent of a swan or a frog, the researchers used a technique called X-ray crystallography. This technique traps the folded protein in a crystal and allows a picture of its shape to be created based on how X-rays bounce off it.

Taking an X-ray snap-shot of two nearby PKCι molecules, Professor McDonald found part of one PKCι was stuck to the other.

This was an unexpected finding and taking a closer look he narrowed down the sticky patch to just four amino acids – when you think PKCι is made up of 596 amino acids, a sticky region of four is quite small.

He named the region RIPR, based on the single letter code used for those four amino acids.

Does RIPR flip the switch?

PKCι sends signals around the cell by tagging other proteins with a marker that changes how that protein behaves.

When the researchers spotted the RIPR motif, the first theory that sprung to mind was that its stickiness might help one PKCι molecule switch on another.

To test this idea, the researchers scrambled the spelling of RIPR by making artificial changes to the DNA recipe for PKCι. These spelling mistakes were designed to make RIPR less sticky so the researchers could test if this affected the day-to-day job of PKCι.

They mixed ‘normal’ PKCι, or the less sticky form, with a chunk of protein – known as a substrate – that PKCι loves to add its tag to.

But when they fired up this reaction, PKCι with the less sticky RIPR was just as good at tagging the substrate as ‘normal’ PKCι.

Dr Mark Linch, one of the lead researchers on the study said this of those puzzling early results: “Although our first theory wasn’t quite right, we were now gripped by the question of what the function of the RIPR motif was. We took a leap of faith, changed our strategy and this led to some fascinating results.”

Sports coach

The researchers knew that for PKCι to do its job, it must be able to grab hold of other proteins. They reasoned that the RIPR motif might provide the grip needed to hold on to other proteins.

By taking a roll-call of what proteins PKCι grabs hold of, the researchers were able to see that two important ones were missing when RIPR was misspelled. The first missing protein was lethal giant larvae 2 (LLGL2) – the name suggests what happens to fruit flies when they don’t make this protein – and the second was myosin X.

Both LLGL2 and myosin X have jobs controlling polarity.

What really intrigued the researchers was that although LLGL2 and myosin X were missing, other known members of the gang were still there.

This suggested that the RIPR motif may be more than just sticky. In fact, it works like a sports coach, selecting the best team of proteins for a particular job – in this case, polarity.

How does a cell know front from back?

In our bodies we need cell polarity to help form the barriers that protect us from the outside world. This often requires specialised cells – called epithelial cells – that lock together like a jigsaw and separate ‘inside’ from ‘outside’.

To test if RIPR team selection affects cell polarity, the researchers used a clever system to grow cells in gel-like surroundings. In cells with normal PKCι, groups of these cells formed hollow spheres – like microscopic bubbles that mimic an epithelial barrier.

But if the cells produced PKCι with a misspelled RIPR they struggled to form a continuous bubble-like barrier and in fact grew as lots of spheres tangled together.

This told the researchers that a sticky RIPR helps a cell tell front from back.

Bubbles of cells (green) make a single barrier (purple) and errors in RIPR tangle the barrier

Spelling mistakes and cancer

An important question that remained was whether the stickiness of the RIPR motif might add to what researchers already know about PKCι and cancer.

Knowing that artificial changes to the RIPR motif messed up cell polarity in the lab, the researchers decided to see if similar changes to RIPR had been seen in cancer samples.

They scanned through a database of DNA changes from human cancer and found that spelling errors in RIPR were the most common mistake seen in PKCι.

They spotted that a particular error had been seen several times – a DNA mistake that meant the first R of RIPR changed to a different amino acid called cystine, or C for short.

To test if the rogue C affected protein team-selection and polarity, the researchers recreated this spelling error in the lab.

Like with the other changes to RIPR they tested, the researchers found that switching to a C also stopped LLGL2 and myosin X from sticking to PKCι.

Even more intriguing, this single spelling mistake confused the spheres of cells grown in the gel-like environment. Just like the artificial changes designed in the lab, this real life cancer-linked change also stopped polarity.

What does this mean for the future?

This is an exciting finding and suggests that the sports coach role played by the RIPR motif could be key in selecting the right team to power a cells internal compass.

Looking to the future, Dr Linch said: “This discovery generates many more questions. Are there cancer-associated spelling mistakes in other polarity proteins? Are there other cancer-associated lines of communication in cells that link up with faulty PKCι and disrupt polarity? Are polarity proteins good targets for new anti-cancer therapies? These are exciting times in the field of polarity in cancer and there is still a lot of work to do.”

We may not have the complete PKCι team-sheet yet, but one thing we do know is that the teams Professors Parker and McDonald have selected know front from back and their head from their toes, which makes them well placed to keep answering these questions.

Nick

Further information

If you want to hear more from the scientists, you can listen to them talking about their research in the Science Signaling podcast.

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